CN114432238A

CN114432238A - ROS response type controlled release ophthalmic preparation and preparation method thereof

Info

Publication number: CN114432238A
Application number: CN202111545875.6A
Authority: CN
Inventors: 高华; 张书平; 牛盼红; 韦超; 吴跃兰; 曾繁星; 刘思金
Original assignee: Affiliated Ophthalmic Hospital Of Shandong First Medical University Shandong Ophthalmic Hospital
Current assignee: Affiliated Ophthalmic Hospital Of Shandong First Medical University Shandong Ophthalmic Hospital
Priority date: 2021-12-16
Filing date: 2021-12-16
Publication date: 2022-05-06
Anticipated expiration: 2041-12-16
Also published as: CN114432238B

Abstract

The invention provides an ROS response type controlled release ophthalmic preparation and a preparation method thereof, belonging to the technical field of biological medicines. The invention adopts ROS response type nano-drug carrier to encapsulate the active ingredients of the eye external-use drug to obtain the eye preparation with controllable release. In the eye inflammation part, the controlled-release eye preparation meets high-content Reactive Oxygen Species (ROS), the wrapping structure of the ROS response type nano-drug carrier is opened, the eye external drug active ingredients are released, after inflammation disappears, the ROS level is reduced, the ROS opening switch is closed, the eye external drug active ingredients are stopped releasing, and therefore the controlled release of the eye external drug active ingredients is achieved. The ROS response type nano-drug carrier can effectively improve the utilization rate of the active ingredients of the externally applied medicine for the eyes, and can reduce or stop releasing after the inflammation of the eyes disappears, thereby avoiding the side effect of the active ingredients of the externally applied medicine for the eyes on the eyes and realizing the safe and effective treatment of the eye diseases.

Description

ROS response type controlled release ophthalmic preparation and preparation method thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an ROS (reactive oxygen species) response type controlled release ophthalmic preparation and a preparation method thereof.

Background

The corneal inflammatory reaction caused by various factors is called keratitis (keratitis), is one of common ophthalmic diseases, and is also one of the main blinding eye diseases in China. The cornea is located at the forefront of the eyeball and is directly contacted with the outside, so that the cornea is easily damaged by microorganisms, trauma and physical and chemical stimulation factors to cause inflammation. Common treatments for corneal inflammation include eye drops, corneal stroma injection, and wearing drug-loaded contact lenses. Among them, corneal stroma injection not only poses the risk of causing ocular trauma and penetration into the anterior chamber, but also may cause endophthalmitis and visual impairment; the drug-loaded contact lens needs to be worn for a long time, which is not beneficial to discharge produced harmful substances into eyes, and meanwhile, the contact lens is also one of the risk factors causing keratitis, and the wearing process also affects the sight line to cause inconvenient life; eye drops are noninvasive treatments and relatively convenient to use, and are a method generally accepted by patients.

The active ingredients of the current externally applied medicine for the eyes commonly used in clinic can not be controllably released, and after the inflammation of the eyes disappears, the active ingredients of the externally applied medicine for the eyes continue to precipitate on the eyes, which can cause the toxic and side effects related to the medicine, such as secondary glaucoma, complicated cataract, corneal epithelial lesion and the like.

Disclosure of Invention

The ROS-responsive nano-drug carrier is adopted to encapsulate the active ingredients of the externally applied eye medicine, so that the controllable release of the active ingredients of the externally applied eye medicine can be realized, the active ingredients of the externally applied eye medicine are reduced or stopped after inflammation disappears, the toxic and side effects of the medicine on eyes are avoided, and the safe and effective treatment of the eye diseases can be realized.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a controllable-release ophthalmic preparation, which adopts ROS reactive oxygen species response type nano-drug carrier to encapsulate active ingredients of an ophthalmic external drug.

Preferably, the ROS-responsive nano-drug carrier comprises a biodegradable saccharide compound modified by amidation modification of ethylene glycol chitosan; the biodegradable saccharide compound has a ROS-responsive group of a carboxyl group.

Preferably, the ROS-responsive group of the carboxyl group comprises one or more of pinacol ester phenylboronate, thioethers, thioketals, selenium bonds and diselenide bonds, tellurium-containing bonds and oxalate bonds.

Preferably, the preparation method of the ROS-responsive nano-drug carrier comprises the following steps:

mixing a biodegradable saccharide compound solution, an ethylene glycol chitosan aqueous solution and a coupling agent, carrying out amidation reaction, dialyzing, and drying the intercepted product of dialysis to obtain the ROS-responsive nano-drug carrier.

Preferably, the temperature of the amidation reaction is 65-75 ℃.

Preferably, the active ingredient of the ophthalmic external application comprises an active ingredient of glucocorticoid type ophthalmic external application and/or an active ingredient of non-hormone type ophthalmic external application.

Preferably, the glucocorticoid drug comprises one or more of fluorometholone, dexamethasone, prednisolone and the like.

Preferably, the non-glucocorticoid eye external use medicine active component comprises one or more of voriconazole, gatifloxacin, moxifloxacin, tobramycin, tacrolimus, rapamycin and cyclosporine.

Preferably, the mass ratio of the ROS-responsive nano-drug carrier to the active ingredients of the externally applied ophthalmic drug is 1: (3-8).

The invention also provides a preparation method of the controlled release ophthalmic preparation, which comprises the following steps:

mixing the ROS response type nano-drug carrier and the active ingredients of the externally applied ophthalmic drug to obtain a controllable-release ophthalmic preparation; the dosage form of the ROS-responsive nano-drug carrier comprises a solution, a gel or an ointment.

The invention provides a controllable release ophthalmic preparation, which adopts ROS response type nano drug carrier to encapsulate active ingredients of an ophthalmic external drug. The invention adopts ROS response type nano-drug carrier to encapsulate the active ingredients of the eye external drug to obtain the controlled-release eye preparation. The eye inflammation part can generate high-content Reactive Oxygen Species (ROS), the eye preparation with controllable release meets the high-content ROS, the structure of the ROS response type nano-drug carrier is opened, the active ingredients of the eye external drug are released, after inflammation disappears, the ROS level is reduced, an ROS opening switch is closed, the release of the active ingredients of the eye external drug is reduced or stopped, so that the controllable release of the active ingredients of the eye external drug is realized, the rest active ingredients of the eye external drug can be stored in the ROS response type nano-drug carrier, under the action of lysozyme, monosaccharide or polysaccharide is generated and absorbed by a human body. The ROS response type nano-drug carrier can effectively improve the utilization rate of active ingredients of the externally applied drug for eyes, avoid the toxic and side effects of the drug and realize safe and effective treatment of eye diseases.

Drawings

FIG. 1 shows the comparison of ROS response performance of GC-EB-DSP and GC nano-drugs;

FIG. 2 shows the results of cell experiments in which GC-EB-DSP releases DSP in response to ROS;

FIG. 3 shows the nano material pair H with different loading concentrations of DSP₂O₂Response performance results of;

FIG. 4 is an infrared characterization of GC-EB materials modifying EB molecules of different ratios.

Detailed Description

The invention provides a controllable release ophthalmic preparation, which adopts ROS response type nano drug carrier to encapsulate active ingredients of an ophthalmic external drug.

In the present invention, the ROS-responsive nano-drug carrier is preferably spherical particles in shape; the particle size of the ROS response type nano-drug carrier is preferably 20-50 nm, and more preferably 30-40 nm.

In the present invention, the ROS-responsive nano-drug carrier preferably comprises a biodegradable saccharide compound modified by amidation modification with ethylene glycol chitosan; the biodegradable saccharide compound has a ROS-responsive group of a carboxyl group. In the invention, the glycol chitosan can self-curl into spherical particles in a solvent system, and the amphiphilic characteristic of the glycol chitosan can realize the entrapment of hydrophobic drugs and hydrophilic drugs at the same time.

In the invention, the ROS response mechanism is dependent on that the ROS response group modified on the surface of the nano material generates bond breakage when meeting ROS, and releases the drug loaded in the interior.

In the invention, the ROS response group of the carboxyl preferably comprises one or more of phenylboronic acid pinacol ester with carboxyl, thioether, thioketal, selenium bond, diselenide bond, tellurium bond and oxalate bond; the biodegradable saccharide compound is preferably one or more of ferrocene, anthocyanin, unsaturated lipid compounds, imidazole compounds and nitroimidazole compounds.

In the present invention, the ROS-responsive group of the carboxyl group preferably comprises pinacol 4-carboxyphenylborate.

In the present invention, the method for preparing the ROS-responsive nano-drug carrier preferably comprises the following steps: and mixing the ROS response group of the carboxyl, the glycol chitosan aqueous solution and the coupling agent, carrying out amidation reaction, dialyzing, and drying the intercepted product of dialysis to obtain the ROS response type nano-drug carrier.

In the present invention, the solvent for the ROS-responsive group of the carboxyl group is preferably methanol; the concentration of the biodegradable saccharide compound in the biodegradable saccharide compound solution is preferably 5-20 mug/mL; more preferably 13.59. mu.g/mL.

In the invention, the concentration of the glycol chitosan in the glycol chitosan aqueous solution is preferably 0.01-1 mug/mL; more preferably 0.417. mu.g/mL.

In the present invention, the coupling agent is preferably N-hydroxysuccinimide (NHS) and (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) (EDC); the mass ratio of the NHS to the EDC is preferably (0.8-1.2): (0.8 to 1.2), more preferably 1:1.

in the invention, the temperature of the amidation reaction is preferably 65-75 ℃, more preferably 70 ℃, and under the temperature condition, the amidation reaction is fast and sufficient; the amidation reaction time is preferably 20-30 h, and more preferably 24 h; the amidation reaction is carried out with stirring.

In the present invention, the dialysis includes a first dialysis and a second dialysis in this order; the dialysate used for the first dialysis is preferably methanol, and the time of the first dialysis is preferably 24 h; the dialysate used for the second dialysis is preferably water; the time of the second dialysis is preferably 48 h.

In the present invention, the drying is preferably freeze-drying; the temperature of the freeze-drying is preferably-50 ℃.

In the present invention, the active ingredient for ophthalmic external preparations includes a glucocorticoid-based active ingredient for ophthalmic external preparations and/or a non-hormone-based active ingredient for ophthalmic external preparations. In the invention, the glucocorticoid medicine preferably comprises one or more of fluorometholone, dexamethasone, prednisolone and the like; the non-glucocorticoid eye external use medicine active component preferably comprises one or more of voriconazole, gatifloxacin, moxifloxacin, tobramycin, tacrolimus, rapamycin and cyclosporine.

In the invention, the mass ratio of the ROS response type nano-drug carrier to the active ingredients of the externally applied medicine for eyes is 1: (3-8), more preferably 1: 5.

In the present invention, the solvent of the aqueous solution of the ROS-responsive nano-drug carrier is preferably distilled water; the concentration of the ROS-responsive nano-drug carrier in the aqueous solution of the ROS-responsive nano-drug carrier is preferably 0.1-1.0 g/L, and more preferably 0.7 g/L.

In the invention, the preparation method of the aqueous solution of the ROS-responsive nano-drug carrier preferably mixes the ROS-responsive nano-drug carrier with water, and then carries out ultrasonic treatment to obtain the aqueous solution of the ROS-responsive nano-drug carrier; the frequency of the ultrasonic wave is preferably 40 KHz; the temperature of the ultrasound is preferably 37 ℃; the ultrasonic time is preferably 9-16 h, and more preferably 12 h; the ultrasound dissolves the ROS-responsive nano-drug carrier in water.

After the aqueous solution of the ROS response type nano-drug carrier is mixed with the active ingredients of the externally applied ophthalmic drug, the method also comprises dialysis and drying of dialysate; the drying is preferably freeze drying.

In the invention, the cut-off molecular weight of a dialysis bag used for dialysis is 2000 daltons; the length of the dialysis bag is preferably 44mm, and the width of the dialysis bag is preferably 45 mm; the dialysate used for dialysis is preferably ultrapure water; the dialysis time is preferably 48 h; the drying mode is preferably freeze drying; the temperature of the freeze drying is preferably-50 ℃; the freeze drying time is preferably 2-3 days.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

0.816g of 4-carboxyphenylboronic acid pinacol Ester (EB) was dissolved in 45mL of methanol, 0.025mg/mL of an aqueous solution of ethylene Glycol Chitosan (GC) (15 mL) was added thereto, the mixture was stirred uniformly, 0.563g N-hydroxysuccinimide (NHS) and 0.761g of (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) (EDC) were added thereto, the mixture was heated to 70 ℃ and the reaction was continued with stirring for 24 hours. After three days of dialysis, GC-EB modified by EB was obtained by freeze-drying.

The infrared spectrum is characterized by being 1725cm^-1New boron ester C ═ O bond appearing after phenylboronic acid modification, and at 714cm^-1At B-O bond, 1544 and 902cm^-1And the C ═ C bond stretching vibration peak of a benzene ring proves that the modification of the phenyl boronic acid used for ROS response type molecules is successful.

Comparative example 1

0.038g of pinacol 4-carboxyphenylborate (EB) was dissolved in 45mL of methanol, added to 15mL of a 2mg/mL aqueous GC solution, stirred well, added with 0.037g N-hydroxysuccinimide (NHS) and 0.036g of (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) (EDC), heated to 70 ℃ and reacted for 24h with continuous stirring. After three days of dialysis, GC-EB modified by EB was obtained by freeze-drying.

Comparative example 2

0.040g of pinacol 4-carboxyphenylborate (EB) was dissolved in 45mL of methanol, added to 15mL of a 2mg/mL aqueous GC solution, stirred well, added with 0.030g N-hydroxysuccinimide (NHS) and 0.036g of (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) (EDC), heated to 70 ℃ and reacted for 24h with continuous stirring. After dialysis for three days, GC-EB modified by EB was obtained by freeze drying.

Comparative example 3

0.202g of pinacol 4-carboxyphenylborate (EB) was dissolved in 45mL of methanol, added to 15mL of 0.05mg/mL GC aqueous solution, stirred well, added with 0.218g N-hydroxysuccinimide (NHS) and 0.200g of (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) (EDC), heated to 70 ℃ and reacted for 24h with continuous stirring. After three days of dialysis, GC-EB modified by EB was obtained by freeze-drying.

Example 2

0.01g of GC-EB prepared in example 1 was dissolved in 15mL of distilled water, sonicated for 15min, and 0.030g of Dexamethasone Sodium Phosphate (DSP) was added to the above solution system, and stirring was continued for 24 h. And (3) carrying out dialysis and freeze drying to prepare the GC-EB-DSP ROS responsive nano-drug carrier.

Example 3

0.01g of GC-EB prepared in example 1 was dissolved in 15mL of distilled water, sonicated for 15min, and 0.050g of Dexamethasone Sodium Phosphate (DSP) was added to the above solution system and stirred continuously for 24 h. And (4) carrying out dialysis and freeze drying to prepare the GC-EB-DSP ROS-responsive nano-drug carrier.

Example 4

0.01g of GC-EB prepared in example 1 was dissolved in 15mL of distilled water, sonicated for 15min, and 0.080g of Dexamethasone Sodium Phosphate (DSP) was added to the above solution system and stirred continuously for 24 h. And (3) carrying out dialysis and freeze drying to prepare the GC-EB-DSP ROS responsive nano-drug carrier.

Example 5

0.01g of GC-EB prepared in example 1 was dissolved in 15mL of distilled water, sonicated for 15min, and 0.010g of Voriconazole (VOR) was dissolved in 2mL of isopropanol-ethanol (1:1), and then added to the above GC-EB solution system, and 3mL of isopropanol-ethanol was additionally added dropwise, and stirring was continued for 24 h. And (3) carrying out dialysis and freeze drying to prepare the GC-EB-VOR ROS responsive nano-drug carrier.

Example 6

0.01g of GC-EB prepared in example 1 was dissolved in 15mL of distilled water, sonicated for 15min, and 0.054g of Voriconazole (VOR) was dissolved in 2mL of isopropanol-ethanol (1:1), and then added to the above GC-EB solution system, and 3mL of isopropanol-ethanol was additionally added dropwise, and stirring was continued for 24 h. And (4) carrying out dialysis and freeze drying to prepare the GC-EB-VOR ROS response type nano-drug carrier.

Example 7

0.01g of GC-EB prepared in example 1 was dissolved in 15mL of distilled water, sonicated for 15min, and 0.100g of Voriconazole (VOR) was dissolved in 2mL of isopropanol-ethanol (1:1), and then added to the above GC-EB solution system, and 3mL of isopropanol-ethanol was additionally added dropwise, and stirring was continued for 24 h. And (3) carrying out dialysis and freeze drying to prepare the GC-EB-VOR ROS responsive nano-drug carrier.

Example 8 comparison of ROS response Performance

1. The results of comparing GC-EB-DSP and GC-DSP (materials with GC material not modified with EB molecules directly encapsulating DSP) and GC nano-drug ROS response performance are shown in FIG. 1.

The concentrations are all 200 mug mL^-1The nanomaterials of GC, GC-DSP and GC-EB-DSP were dispersed in H at different concentrations₂O₂Solution (0,2.5,5, 10. mu.g mL)^-1) After stirring for 24h, the released drug was tested. GC and GC-DSP materials have no obvious ROS response performance; GC-EB-DSP with H₂O₂The addition concentration is increased, the DSP drug release concentration is increased, and obvious ROS response performance is shown.

2. GC-EB-DSP cell experiments releasing DSP in response to ROS. Lipopolysaccharide (LPS, 1ug mL) for corneal epithelial cells^-1) ROS production was stimulated as a Positive control (Positive), and cells without LPS stimulation and added nanomaterial as a negative control (Ctrl). The experimental components stimulated by LPS are respectively added with 200 mug mL^-1After the GC-EB and GC-EB-DSP nano materials are treated overnight and PBS is washed for three times, the ROS detection kit is adopted to image the fluorescent staining in the cells through laser confocal. As shown in fig. 2, GC-EB material showed a response-clearing effect on ROS after modification of EB molecules, compared to control group, and thus green fluorescence of ROS kit disappeared; the response clearance effect on ROS is still shown after the DSP medicament is encapsulated. Therefore, the prepared GC-EB-DSP shows good ROS response performance.

3. Nano material pair H for verifying different entrapment concentrations of DSP₂O₂See figure 3 for response performance results.

Encapsulating nano-materials prepared from DSPs with different concentrations at different concentrations H₂O₂(0,2.5,5,10μg mL^-1) The concentration of released DSP varies under the stimulation conditions. When the concentration of DSP added is 50mg mL^-1The prepared GC-EB-DSP-50 showed the best H₂O₂A response capability.

4. GC and EB starting materials (1:0.5,1:1.5 and 1:20) in different reaction ratios were passed through 0.218g N-hydroxysuccinimide (NHS) and0.200g of (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) (EDC) was heated to 70 ℃ and the reaction was continued with stirring for 24 h. After three days of dialysis, GC-EB products modified with EB molecules of different contents were obtained. When the molar concentration of GC to EB is 1:20, an optimized GC-EB ROS response type nano-drug carrier is obtained, and the result is shown in figure 4. As can be seen from FIG. 4, the infrared spectrum was characterized at 1725cm at a GC to EB molar concentration of 1:20^-1New boron ester C ═ O bond appearing after phenylboronic acid modification, and at 714cm^-1At B-O bond, 1544 and 902cm^-1And the C ═ C bond stretching vibration peak of the benzene ring proves that the modification success of the phenyl boronic acid used for the ROS responsive molecule is realized, and the optimized GC-EB ROS responsive nano carrier is obtained.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A controllable release ophthalmic preparation adopts ROS response type nano drug carrier to encapsulate active ingredients of an ophthalmic external drug.

2. The ophthalmic formulation of claim 1, wherein the ROS-responsive nano-drug carrier comprises biodegradable saccharide compounds modified by amidation with ethylene glycol chitosan; the biodegradable saccharide compound has an ROS-responsive group; the ROS-responsive group is a carboxyl ROS-responsive group.

3. The ophthalmic formulation of claim 2, wherein the ROS-responsive group of the carboxyl group comprises one or more of pinacol ester phenylboronate, thioethers, thioketals, selenium linkages, diselenide linkages, tellurium-containing linkages, and oxalate linkages.

4. The ophthalmic formulation according to claim 2 or 3, characterized in that the preparation method of the ROS-responsive nano-drug carrier comprises the following steps:

5. The ophthalmic formulation according to claim 4, characterized in that the temperature of the amidation reaction is 65-75 ℃.

6. The ophthalmic formulation according to claim 1, characterized in that said ophthalmic external drug active ingredient comprises a glucocorticoid based ophthalmic external drug active ingredient and/or a non-hormonal based ophthalmic external drug active ingredient.

7. The ophthalmic preparation of claim 6, wherein the glucocorticoid drug comprises one or more of fluorometholone, dexamethasone, prednisolone, etc.

8. The ophthalmic formulation according to claim 6, wherein the non-glucocorticoid ophthalmic external application active ingredient comprises one or more of voriconazole, gatifloxacin, moxifloxacin and tobramycin, tacrolimus, rapamycin and cyclosporine.

9. The ophthalmic preparation as claimed in claim 1, wherein the ratio of ROS responsive nano drug carrier to the active ingredient of the external ophthalmic drug is 1: (3-8).

10. A process for the preparation of a controlled release ophthalmic formulation according to any one of claims 1 to 9, comprising the steps of: